1. Lab (3): Liver Function profile (LFT) Lecturer Nouf Alshareef KAU-Faculty of Science- Biochemistry department Clinical biochemistry lab (Bioc 416) 2012 noalshareef@kau.edu.sa

2. Liver Function Test (LFT) profile Synthetic functionExecratory function Integrity of liver cells Bilirubin Bilirubin, ALP Proteins TP, Alb, A/G ratio Liver enzymes AST, ALT, GGT, ALP

3. What is Bilirubin ??? Is water insoluble compound produced in spleen from heme degradation. [Heme: iron + organic compound “porphyrin”]. Heme source in body: 80% from hemoglobin, 20% other hemo-protein: cytochrome, myoglobin)

4. Structure Heme four pyrrols rings connected together to form (porphyrin). Bilirubin consists of open chain of four pyrrols-like rings

5. Hemoglobin degrading and bilirubin formation Spleen Plasma Protein and a.a pool Iron pool TO LIVER Liver globin iron Heme Hemoglobin Bilirubin Binds with albumin Conjugation process

6. In liver: Bilirubin is conjugated with Glucouronic acid to produce bilirubin diglucuronides, which is water soluble and readily transported to bile. Blilirubin + Glucouronic acid bilirubin diglucuronides Then conjugated bilirubin is excreted in bile through bile duct to help in food digestion. The excess amount transferred to intestine to be excreted in urine and stool. UDP-glucuronyl transferase "water soluble" "Conjugated BIL" "water in soluble" Bile

8. Elevated levels of bilirubin in blood and urine indicate certain diseases.

9. Direct bilirubin: is conjugated (water soluble bilirubin) in aqueous solution it reacts rapidly with reagent (direct reacting). Indirect bilirubin: is unconjugated (water insoluble bilirubin) because it is less soluble in it reacts more slowly with reagent (reaction carried out in methanol). - in this case both conjugated and unconjugated bilirubin are measured given total bilirubin. Unconjugated will calculated by subtracting direct from total and so called indirect. Total bilirubin = D+ ID Knowing the level of each type of bilirubin has diagnostic important.

11. Post-hepatic jaundiceHepatic jaundicePre-hepatic jaundice  Due to obstruction of bile duct which prevents passage of bilirubin into intestine.  D.Bil will back to liver and then to circulation elevating its level in blood and urine.  Due to liver cell damage (cancer, cirrhosis or hepatitis)  Conjugation of bilirubin decreased (ID.Bil.  ).  Blilirubin that is conjugated is not efficiently secreted into bile but leaks to blood (D.Bil.  )  Due to increase in RBCs breakdown due to hemolytic anemia.  The rate of RBCs lysis and bilirubin production more than ability of liver to convert it to the conjugated form. Cause D.Bil (High) D.Bil, ID.Bil, T.Bil all (High) ID.Bil > D.BilType of Bil. ALP ( High)ALT, AST (High) K + ( High) Hematology: CBC (low Hb) Conformational test haemolytic jaundice hepato-cellular jaundiceobstructive jaundice

12. High bilirubin levels is common in newborns age (1-3 days). After birth the newborns breaking down the excess RBCs they are born with and, because the newborn’s liver is not fully mature, (unable to process the extra bilirubin) leads to elevate its level in blood and other body tissues. This situation usually resolves itself within a few days. SO, WHAT TYPE OF JUNDUCE IS THIS ???

13. Usually newborn is treated by phototherapy which breakdown bilirubin (ID  D) and convert it to the photo isomer form which is more soluble. Very high bilirubin is danger and toxic it may cause brain damage and effect on muscles, eyes and leading to death.

14. Experiment: Measuring serum bilirubin level Principle: Sulphanalic acid + NaNO3 diazotized sulphanalic acid (DSA) DSA + Bilirubin “D” Azobilirubin “purple” Bilirubin “ID”+ DSA + accelerator Total bil. (methanol )

15. Kit components Reagents: Sulfanalic acid reagent Sodium nitrate reagent Methanol reagent Bilirubinequavalent standard (5mg/dl T.bil; 2.5 mg/dl D.bil)

16. Procedure: TestTest blank 1.4 ml Sulfanilic acid 25  l -NaNO 3 - 25  l dis. H 2 O Mix, stand for 1 min 100  l Sample - Read the Abs after 1 min at 540nm, (Blank dis. H 2 O) Use this to calculate D.bil 1.5 ml Methanol - Mix by inversion, stand 5 min or more - Read Abs. at 540nm (Blank dis. H 2 O) Use this to calculate T.bil - Pour Bilirubinequavelant standard in clean cuvette read Abs. at 540nm, (blank dis. H 2 O)

17. C C alculations Conc. of Bilirubinequavelant is 5mg/dl for T.bil, and 2.5 mg/dl for D.bil D.Bil: Abs (test) - Abs (test blank) X 2.5 mg/dl T.Bil: Abs (test) - Abs (test blank) X 5 mg/dl To convert to  mol/L multiply by 17.1 Abs of Bilirubinequavelant

18. Normal range D.Bil: 0.5 mg/dl or 8.6  mol/L T.Bil: 1 mg/dl or 17.1  mol/L